Robert J. Plunkett

Neural stem cell detection, characterization, and age-related changes in the subventricular zone of mice.

The mammalian brain contains neural stem cells (NSCs) that allow continued neurogenesis throughout the life of the animal. However, neurogenesis is known to decline during aging and, to the extent that neurogenesis is required for normal CNS function, this may contribute to neurodegenerative disease. Decreased neurogenesis could result from loss of NSCs or dysfunction at some later step, and distinguishing these possibilities is important for understanding the cause of the decline. However, because of the inability to distinguish NSCs from their rapidly dividing progeny in situ, it has not been possible to quantitatively assess the NSC populations in young and old animals. In this report we show that the G1 phase-specific expression of the replication factor Mcm2 is a useful marker for detecting slowly cycling putative NSCs in situ and confirm the identity of these cells using both cytosine β-d-arabinofuranoside (Ara-C) treatment and a double nucleoside analog-labeling technique. The ability to distinguish NSCs from proliferative progenitors has allowed characterization of the expression of several markers including Nestin, Musashi, and GFAP in these different cell types. Furthermore, comparison of the NSC populations in the subventricular zones of young (2-4 months) and old (24-26 months) mice demonstrates an approximately twofold reduction in the older mice. A similar twofold reduction is also observed in the number of neurospheres recovered in culture from old relative to young animals. The reduction in the neural stem cell population documented here is sufficient to account for the reduced level of neurogenesis in old animals.

Correlation of Endothelin-1 and Transforming Growth Factor β1 with Malignancy and Vascularity in Human Gliomas

Because the prominent neovascularization characteristic of high grade primary brain tumors is composed mostly of vascular smooth muscle cells (VSMC), we studied the expression of the potent smooth muscle mitogen endothelin-1 (ET-1) and one of its secretagogues, transforming growth factor β1 (TGF-β1) in a series of astrocytic tumors. TGF-β1 is also of interest due to its known activity as an angiogenic factor. Using immunohistochemical methods, we examined 30 surgical cases: 10 glioblastoma multiforme, 10 anaplastic astrocytomas, and 10 low-grade astrocytomas. Using a monoclonal antibody to TGF-β1 and a polyclonal antibody to ET-1, we detected both growth factors in all cases of glioblastoma examined. In cases of anaplastic astrocytoma, 4 tumors were positive for both factors; 2 contained only ET-1; 2 contained only TGF-β1; and 2 exhibited no tumor cell immunoreactivity for either factor. In low-grade astrocytoma, 4 of 10 tumors showed weak ET-1 immunoreactivity; 2 of those contained TGF-β1 immunopositive tumor astrocytes; 6 tumors were negative for both factors. In all tumors that expressed both factors, serial sections showed that regions of ET-1 immunopositivity also tended to be positive for TGF-β1. Endothelial cells within all tumors were positive for ET-1. ET-1 and TGF-β1 are present in human astrocytomas and their expression correlates with tumor vascularity and malignancy. These results suggest roles for both ET-1 and TGF-β1 in the growth and progressive angiogenesis of the human glioma.

Chapter 70 Transient behavioral recovery in hemiparkinsonian primates after adrenal medullary allografts

Publisher Summary This chapter describes and examines resulting changes in volitional and drug-induced motor function after an interval of six months using hemiparkinsonian monkeys as recipients for adrenal medullary allografts. The results indicate that adrenal medullary allografts into the caudate nucleus of l-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridin (MPTP) hemiparkinsonian monkeys affect transient improvement in function of the basal ganglia as measured by volitional arm use and reduction in apomorphine-induced turning activity, which is apparent three months after implantation. The transient improvement in motor performance after adrenal medullary implants may be attributed to early survival of catecholamine-producing cells and release of dopamine from the chromaffin cells. Placement of a tissue implant, whether adrenal medulla, cortex, or fat, or the creation of a surgical wound in the head of the caudate, appears to cause dopamine fiber growth from intact dopaminergic neurons into the damaged caudate nucleus. The chapter suggests that implantation of fetal tissue into the caudate induces sprouting from the ventral striatum and can produce bilateral functional improvement in monkeys with the full parkinsonian syndrome after intravenous MPTP, and in hemiparkinsonian monkeys.

Thiopental Modification of Ischemic Spinal Cord Injury in the Dog

Spinal cord ischemia was produced in male mongrel dogs by permanent occlusion of the infrarenal aorta. All animals were anesthetized with a mixture of nitrous oxide and 1.5% halothane. Group 1 animals were the controls. Group 2 animals were pretreated, 30 minutes prior to aortic occlusion, with sodium thiopental, 20 mg per kilogram of body weight, over 5 minutes, followed by an infusion of 10 mg/kg/hr for 2 1/2 hours. Groups 3 animals received the identical dose of sodium thiopental and, in addition, received mannitol, 1 gm/kg, and methylprednisolone 1 mg/kg. There were no differences in hemodynamic data or arterial blood gases among the groups, except that the thiopental bolus caused a transient reduction in mean arterial pressure. Ninety percent of Group 1 animals were paraplegic, while only 30% of Group 2 and 40% of Group 2 animals were paraplegic. The difference in the incidence of paraplegia in Groups 2 and 3 compared with Group 1 was statistically significant (p less than 0.05). Therefore, thiopental significantly decreased the incidence of paraplegia, while methylprednisolone and mannitol did not enhance its protective effect.

Chapter 63 Behavioral recovery from MPTP-induced parkinsonism in monkeys after intracerebral tissue implants is not related to CSF concentrations of dopamine metabolites

Publisher Summary l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) administered to primates causes the rapid development of a movement disorder that closely resembles Parkinsons disease. The toxicity of MPTP depends on its conversion by monoamine oxidase B to its pyridinium derivative, MPP +, which is concentrated selectively in catecholaminergic neurons. Data presented in the chapter are the part of a multidisciplinary study of MFTP effects and the ability of implanted tissue grafts to reverse the MPTP-induced motor deficits in nonhuman primates. The survival of fetal mesencephlic tissue implanted into the primate neostriatum and at least a partial reversal of the MPTP-induced motor deficits has been demonstrated in monkeys. Surviving implanted dopaminergic neurons can synthesize, store, and release dopamine; they also can form processes that are believed to innervate the damaged host neostriatum. The implantation of nondopaminergic fetal amniotic tissue also elicits behavioral recovery. The behavioral recovery in MPTP-hemiparkinsonian monkeys after fetal dopaminergic implants is a consequence of stimulation of growth of host dopaminergic neuronal sprouts rather than the result of secretion of dopamine by the graft or ingrowth of fibers from the dopamine-rich implant. The chapter examines whether biochemical alterations in the cerebrospinal fluid (CSF) reflect the apparent changes in dopaminergic function, which attend the development of hemiparkinsonism in rhesus monkeys after a unilateral intracarotid artery infusion of MPTP and after the implantation of various tissue grafts that produce transient or permanent functional motor improvement in these animals.

Recovery in hemiparkinsonian rats following intrastriatal implantation of activated leukocytes

Behavioral improvement has been seen in hemiparkinsonian animals after surgical lesions of the denervated caudate nucleus. This study was designed to investigate the role of inflammatory cells in injury-induced recovery. A hemiparkinsonian syndrome was induced in rats by unilateral injections of 6-hydroxydopamine into the pars compacta of the substantia nigra. Phytohemagglutinin-stimulated rat peritoneal cells, predominantly T cells and macrophages, were stereotactically implanted in the lesioned caudate-putamen, and amphetamine-induced turning was used to assess recovery. Animals receiving implants of activated peritoneal cells showed a 47% decrease in amphetamine-induced turning 8 weeks after implantation, which was not seen in control or sham-operated animals. Immunocytochemistry revealed increased tyrosine hydroxylase reactive fibers in the leukocyte-implanted striatum. We conclude that implantation of activated leukocytes promotes functional recovery in hemiparkinsonian rats.

Intrathecal baclofen in multiple sclerosis: Too little, too late?

The majority of patients with multiple sclerosis (MS) have symptoms of spasticity that increasingly impair function as the disease progresses. With appropriate treatment, however, quality of life can be improved. Oral antispasticity medications are useful in managing mild spasticity but are frequently ineffective in controlling moderate to severe spasticity, because patients often cannot tolerate the adverse effects of increasing doses. Intrathecal baclofen (ITB) therapy can be an effective alternative to oral medications in patients who have a suboptimal response to oral medications or who cannot tolerate dose escalation or multidrug oral regimens. ITB therapy may be underutilized in the MS population because clinicians (a) are more focused on disease-modifying therapies rather than symptom control, (b) underestimate the impact of spasticity on quality of life, and (c) have concerns about the cost and safety of ITB therapy. Delivery of ITB therapy requires expertly trained staff and proper facilities for pump management. This article summarizes the findings and recommendations of an expert panel on the use of ITB therapy in the MS population and the role of the physician and comprehensive care team in patient selection, screening, and management.

Syngeneic central nervous system transplantation of genetically transduced mature, adult astrocytes.

Advances in the development of highly infectious, replication-deficient recombinant retroviruses provide an efficient means of stable transfer of gene expression. Coupled with ex vivo transduction, surrogate cell populations can be readily implanted into the brain, thus serving as vehicles for delivering selected gene products into the central nervous system (CNS). Here we report that rat astrocytes can be routinely and safely isolated from brain tissue of a living donor by use of short-term gelatin sponge implants. The mature, nontransformed astrocytes were easily expanded, maintained in long-term tissue cultures and were efficiently transduced with an amphotropic retrovirus harboring a heterologous, fused transgene. In vitro retroviral infection rendered the nontransformed cells essentially 100% viable after exposure. The level of efficiency of infection (30–50% effective genome integration of provirus and expression of transgene in target cell populations) and minimal cell toxicity obviated the need to harvest large numbers of target cells. Cultured transduced astrocytes were resilient and exhibited select peptide expression for up to 1 year. Subsequently, transduced astrocytes were used in a series of experiments in which cells were transplanted intracerebrally in syngeneic animals. Post-implantation, astrocytes seeded locally and either insinuated into the surrounding parenchyma in situ or exhibited a variable degree of migration, depending on the anatomic source of astrocytes and the targeted brain implantation site. Transduced astrocytes remained viable in excess of 8 months post-transplantation and exhibited sustained transgenic peptide expression of green fluorescent protein/neomycin phosphotransferase in vivo. The sequential isolation and culture of nontransformed, mature, adult astrocytes and recombinant retrovirus-mediated transduction in vitro followed by brain reimplantation represents a safe and effective means for transferring genetic expression to the CNS. This study lays the foundation for exploring the utility of using a human autologous transplantation system as a potential gene delivery approach to treat neurological disorders. Prepared and utilized in this manner, autologous astrocytes may serve as a vehicle to deliver gene therapy to the CNS.

Estrogen increases survival in an orthotopic model of glioblastoma

Despite the male preponderance for developing glial tumors and a body of published literature that suggests a female gender advantage for long term survival in both human and animal studies, there have been relatively few rigorous investigations into the hormonal effects on glial tumor growth. In a previous study, we concluded that estrogen played a major role in the female survival bias seen in an intracerebral nude rat model of glioblastoma multiforme. Here we explore the potential therapeutic effect of exogenous estradiol delivery in nude rats with orthotopic glioblastoma tumors and examine the mechanism of action of estradiol on reducing tumor growth in this animal model. We administered estradiol, in several dosing regimens, to male, female and ovariectomized nude rats in a survival study. Brain sections, taken at various time points in tumor progression, were analyzed for estrogen receptor protein, proliferative index and apoptotic index. Estradiol increased survival of male, female and ovariectomized nude rats with intracerebral U87MG tumors, in a gender specific manner. The estradiol mediated effect occurred early in tumor progression, and appeared to be caused in-part by an increase in apoptotic activity. It remains unclear if estradiol’s effect is direct or indirect and if it is estrogen receptor mediated. Estradiol-based or adjunctive therapy may be beneficial in treating GBM and further study is clearly warranted.

Intratumoral infusion of topotecan prolongs survival in the nude rat intracranial U87 human glioma model

Topotecan is a topoisomerase (topo) I inhibitor with promising activity in preclinical studies. We hypothesized that low-dose intratumoral delivery of topotecan would be highly effective for gliomas. Human glioma cell lines (U87, U138 and U373) displayed different sensitivities to topotecan (IC50 range: 0.037 μM to 0.280 μM) in cell culture. The most resistant of the glioma cell lines (U87) was implanted stereotactically into the brains of nude rats. Twelve days later, at which time tumor diameter measured 2 to 2.5 mm, animals were randomized to three groups: group I, intratumoral topotecan infused via osmotic pump (n = 12); group II, intratumoral saline infusion (n = 7); and group III, no treatment (n = 10). Animals were sacrificed when signs of deterioration developed, or at 60 days. Animals in group I had a mean survival time (MST) of > 55 days (range=40–60); whereas, those in groups II and III had MST of 26.1 (range=21–31) and 26.5 (range = 20–30) days, respectively. The differences in survival between group I and each of the other groups were statistically significant (p < 0.0001; Logrank Mantel-Cox). None of the animals that survived 60 days had histological evidence of residual tumor at sacrifice. Measurement of topotecan levels in normal brain revealed cytotoxic concentrations up to 4.5 mm from the site of infusion. This study demonstrates that intratumoral topotecan delivered via an osmotic pump prolongs survival in the U87 human glioma model.